Background: Paying attention to their patients’ work and recognizing work-related problems is challenging for many general practitioners (GPs).Objectives: To assess the effect of training designed to improve the care for patients with work-related problems in general practice.Methods: A cluster randomized controlled trial among 32 Dutch GPs. GPs in the intervention group received five-hour training. GPs in the control group were not trained. Included patients  completed baseline questionnaires and follow-up questionnaires planned after one year. Primary outcome at patient level was patients’ expectations about their ability to work, measured using the return-to-work self-efficacy scale (RTW-SE). Primary outcomes on GP level were their use of ICPC-code Z05 (‘work-related problem’) per 1000 working-age patients and percentage of the electronic medical files of working-age patients in which information about occupation had been recorded.Results: A total of 640 patients completed the baseline questionnaire and 281 the follow-up questionnaire. We found no statistically significant differences in patients’ RTW-SE scores: intervention 4.6 (95%CI: 4.2–5.0); control 4.5 (95%CI: 4.1–4.9). Twenty-nine GPs provided data about the GP-level outcomes, which showed no statistically significant differences: use of ICPC code Z05 11.6 (95%CI: 4.7–18.6) versus 6.0 (95%CI: –1.2 to 13.2) per 1000 working-age patients; recording of occupation 28.8% (95%CI: 25.8–31.7) versus 28.6% (95%CI: 25.6–31.6).Conclusion: Training GPs did not improve patients’ work-related self-efficacy or GPs’ registration of work-related problems and occupation. KEY MESSAGESTraining GPs did not improve patients’ expectations concerning their ability to work.Training GPs did not increase recording of occupation and use of ICPC-code Z05.Educational training for GPs based on GPs’ needs, including personalized feedback, might improve care for patients with work-related problems.Work is important for a person’s health and wellbeing ,2. Work-GPs, trusted by their patients and working at the point of entry into the healthcare system, are considered to be ideally positioned to quickly recognize WRPs and prevent avoidable sickness absence . TherefoMany strategies primarily aimed to reduce sick leave but one can argue whether this should be the main focus. That many people with serious health problems continue working illustrates how vital work can be for health and wellbeing ,15. PeopTraining GPs can positively influence their performance and result in beneficial effects for patients. This was demonstrated in studies on domestic violence, advice on smoking cessation, and management of lower respiratory tract infections . We assuThis study was a cluster RCT among Dutch GPs about the effect of training to improve GPs’ care for patients with WRPs. The minimum follow-up time was one year. Each participating practice (one or more GPs) was a cluster. Participation of patients involved the completion of two questionnaires. The study was registered in ‘The Netherlands Trial Registry’ (number NTR3475) and its protocol published in 2014 .In 2011, we sent a letter to all 1400 GPs working in the south-eastern part of the Netherlands inviting them to participate. As only six GPs had responded positively, personally approaching GPs through our networks resulted in 26 more GPs willing to participate. All worked more than two days per week as a GP.Patients were invited to participate by the receptionists if they visited the GP during the study period (17 February 2012 to 31 January 2013). The receptionists asked them to fill out a form checking the inclusion criteria. The criteria were: age 18–63 years; paid work for at least 12 h per week; ability to read and complete questionnaires.Randomization took place at practice level to prevent contamination. The researcher was blinded and matched a random allocation sequence (block size 2) with a randomly ordered list of participating practices. If more than one GP in a practice participated, we informed them about the condition they were allocated to, after all had confirmed participation.The first training session took place on 16 February 2012 and lasted five hours. It consisted of lectures, small group discussions, and role-play. It covered six topics: (1) the connection between work and health; (2) usual care for WRPs and ways to improve it; (3) legislation regarding absenteeism and collaboration with OP; (4) gender aspects of work and WRPs; (5) activating care for patients with work-related distress; (6) registration of occupation and WRPs in the electronic medical record (EMR). The programme was based on the results of a focus group study, in which we found that lack of knowledge and counselling skills were barriers for GPs to identify and address WRPs proactively . The proPrimary patient-level outcome: work-related self-efficacy. Self-efficacy is a measure of a person’s expectations about his or her ability to execute a course of action [f action . Patientf action .Primary GP-level outcomes: recording of WRP and occupation. We used GPs’ use of the ICPC-code ‘Z05’ (for ‘work-related problem’) and their recording of information about occupation in the EMR as primary GP-level outcomes.Secondary GP level outcome: GP work-awareness. We developed the ‘GP work-awareness scale’ (GWAS), to assess patients’ perception of their GP’s attention for work and WRPs. It ranges from 0–4 and is calculated by adding the values of the following four items : (1) ‘does your GP know your occupation?’ (2) ‘did your GP discuss the possibility that your health problem is work-related?’ (3) ‘did your GP discuss sick leave?’ (4) ‘did your GP help finding solutions for WRP?’Other variables. GPs’ experience; the following patient-reported characteristics (supplementary Table): work-relatedness of health problem; educational level; sector of employment; type of contract; working hours/week; monthly  income; experienced health; presence of chronic illness; sick leave; visits to GP; visits to OP. The outcomes were described in more detail in the study protocol [protocol . Not menPatient questionnaires. We assessed the RTW-SE, the GWAS and the ‘other variables’ using questionnaires. The receptionists asked patients meeting the inclusion criteria and consenting to participate, to fill out the baseline questionnaire (paper or online). We used LimeSurvey  software to process the questionnaire data. Twelve months after completion of the baseline questionnaire, research assistants invited all patients of whom addresses were available to fill out an identical follow-up questionnaire (paper or online), also the patients who had not completed the baseline questionnaire.We assessed the primary outcomes on GP level using data extracted from the EMR of the participating GPs during two equal length periods (98 weeks) before and after the intervention. We assessed the use of ICPC-code Z05 by dividing the number of patients for whom this code was used during the pre- and post-periods, by the total number of working-age patients on the GP’s list, expressing the result as number per 1000. We assessed registration of occupation by taking a stratified random sample of 40 patients , who had visited the GP during the post-intervention period. It was positive if at least one of six details about the patient’s occupation had been recorded: (1) employer; (2) sector; (3) occupation; (4) level; (5) working hours; (6) type of contract. We calculated the percentage of the sampled patients for whom registration of occupation was positive.We assumed that we had to be able to demonstrate a moderate effect, defined as half a standard deviation (SD) of the RTW-SE, with a power of 80%  = 0.15). In an earlier study, the mean score of the RTW-SE was 4.24 and the SD 1.14 . Allowint-tests and chi-square tests to assess differences between patients of both study groups. We also compared patients who completed both questionnaires with patients who were lost to follow-up. We calculated the ICC for the RTW-SE at baseline. To assess the effect of the intervention on the RTW-SE at follow-up, we used a multilevel analysis of covariance (ANCOVA) to calculate estimated marginal means [P ≤0.25) with the RTW-SE: patient’s gender; age; educational level; sector of employment; type of contract; monthly income; weekly working hours; experienced health; chronic illness; sick leave; visits to GP; GP’s gender, GP’s experience.The significance threshold was set at 0.05. We used al means . We corrFor each GP, we assessed the use of ICPC-code Z05 and recording of occupation during the pre- and post-intervention period, and calculated the aggregated mean GWAS-score at baseline and follow-up. To estimate the effect of the intervention we used ANCOVA. Post-intervention and follow-up scores were dependent variables, study condition independent variable and post-intervention and baseline scores were covariates . Data waGeneral practitioners. Thirty-two GPs working in 26 practices agreed to participate in our study (intervention (I): 7 female (F)/9 male (M); control (C): 8F/8M. The mean number of years since qualification as GP was 17.0 . Two GPs withdrew from the study after the baseline measurement but one of them later permitted us to extract GP-level data from the EMR. Three other GPs, who continued the study, did not give this permission but one provided us with self-extracted ICPC-code Z05 data. For five GPs, no GWAS data were available at baseline or at follow-up. Therefore, we could analyse data on the use of ICPC code Z05 (WRPs) for 29 GPs, on registration of occupation for 28 GPs, and GWAS-data for 27 GPs.Patients. In total 1306 patients were enrolled; 640 completed the questionnaire at baseline; 281 at baseline and follow-up; 113 only at follow-up. Details are described in P <0.01); more were in the category low (15.4% vs 9.6%) or high education (34.5% vs 29.9%) (P = 0.02); more were self-employed (11.9% vs 6.5%) or temporarily employed (16.7% vs 11.8%) (P <0.01); had a monthly income between €1000 and €2000 (48.2% vs 40.6%) or did not report their income (11.9% vs 8.3%) whereas fewer earned between €2000 and €3000 (15.9% vs 25.5%) (P = 0.02).The mean RTW-SE was the same in both groups at baseline and at follow-up . The ICC was 0.014. The estimated marginal means at follow-up was: intervention: 4.6 (95% CI: 4.2–5.0); control: 4.5 (95% CI: 4.1– 4.9) .P = 0.26).We found no statistically significant differences between the study groups . Use of P = 0.1).The difference between the GWAS score of GPs in both groups was not significant  were that they were targeted to the GPs’ individual needs and provided more opportunities to discuss barriers ,19,24. POne-third of the patients indicated their health problem was possibly work-related. This prevalence is congruent with other studies. A recent French primary care based study found a prevalence of work-related common mental health problems among working-age patients of 26% and a systematic review reported similar numbers ,15. InsiApart from individualizing the training and providing feedback, there is evidence that combining interventions may lead to better results. For example, a combination of peer group supervision, individualized telephone consultations, self-rating scales for professionals, and ongoing measurement of symptom severity in patients resulted in improved uptake of guidelines for the treatment of anxiety and depression by GPs .To our knowledge, this is the first RCT studying the effect of training to improve GPs’ registration of WRPs and their care for patients with work-related problems. Recruiting working-age patients, regardless of the presence of WRPs, enabled us to estimate the work-related self-efficacy and prevalence of WRPs in the (Dutch) GPs’ waiting room population and to be informed about their opinion on GPs’ role in this field. A further strength was that we used outcome measures on both GP and patient levels, and that we assessed patients’ perception of GPs’ management of WRPs.Several limitations may have contributed to the negative result of our trial. First, it was underpowered due to failure to include sufficient numbers of patients and loss to follow-up. Second, not limiting our study to patients experiencing WRPs possibly decreased the necessity for GPs to pay attention to work. This also resulted in a sample with a high baseline RTW-SE, limiting the room for improvement. Third, the outcome measures on a GP level may not reflect other changes in consultations occurring due to the training. Another limitation of the study is that the GWAS scale had not been validated.Given the prevalence of WRPs among patients visiting their GP and the role patients see for GPs, we consider it relevant to study the care of GPs for patients with WRPs. In light of the demonstrated effectiveness of educational interventions in other fields, we recommend development of an intervention aimed at high-risk patients, tailored to the individual needs of the GP, and providing ongoing feedback.Training GPs did not increase patients’ work-related self-efficacy or GPs’ recording of WRPs or occupation; neither did patients experience more attention for work from GPs who were trained.


Proteins have the potential to serve as nanomachines with well-controlled structural movements, and artificial control of their conformational changes is highly desirable for successful applications exploiting their dynamic structural characteristics. Here, we demonstrate an experimental approach for regulating the degree of conformational change in proteins by incorporating a small-molecule linker into a well-known photosensitive protein, photoactive yellow protein (PYP), which is sensitized by blue light and undergoes a photo-induced N-terminal protrusion coupled with chromophore-isomerization-triggered conformational changes. Specifically, we introduced thiol groups into specific sites of PYP through site-directed mutagenesis and then covalently conjugated a small-molecule linker into these sites, with the expectation that the linker is likely to constrain the structural changes associated with the attached positions. To investigate the structural dynamics of PYP incorporated with the small-molecule linker (SML-PYP), we employed the combination of small-angle X-ray scattering (SAXS), transient absorption (TA) spectroscopy and experiment-restrained rigid-body molecular dynamics (MD) simulation. Our results show that SML-PYP exhibits much reduced structural changes during photo-induced signaling as compared to wild-type PYP. This demonstrates that incorporating an external molecular linker can limit photo-induced structural dynamics of the protein and may be used as a strategy for fine control of protein structural dynamics in nanomachines. Various nanomachines based on small-molecule moieties have been designed and synthesized to control structural changes and mimic numerous mechanical movements at atomic-length scales ,2. HowevIt has been reported that the structural changes of photo-responsive organic molecules bound to photo-insensitive proteins can induce subsequent structural changes in the protein. In this way, photo-insensitive proteins can be turned into photoactive proteins. A well-known example is the control of protein structural changes using azo-benzene derivatives . In the p-coumaric acid (pCA) as the chromophore, is an attractive model system for understanding photoreception and the subsequent signal transduction process responsible for the negative phototaxis in Halorhodospira halophila . P. PP(r), Next, we performed shape reconstruction using the DAMMIF program and GNOM data to estimate the shapes of the dark and light states of SML-PYP  22]. Th. Th22]. -1 and produced a model based on all available data to facilitate higher resolution of the protein shape. As shown in Rg values and reconstructed shapes between SML-PYP and wild-type PYP  of SML-PYP by 0.9 Å compared with that of wild-type PYP, which corresponds to an approximate volume increase of 5500 Å3. The volume of the six residues in the N-terminal region would be about 147 Å3 plus the volume of the hydration shell and thus this alone cannot account for the 0.9 Å increase in Rg value. On the other hand, the environment around the chromophore region of SML-PYP is expected to be well conserved and similar to that of the wild-type based on the similarity of the UV-vis spectra  is quite similar to that of wild-type PYP, whereas the CD spectrum of SML-PYP indicates that its α-helix content is moderately reduced . The SAXctra see . The red SML-PYP a. The mo SML-PYP d. The prRg value from 14.4 to 15.6 Å for wild-type PYP upon blue light irradiation . Af. Af2), ahods see .TA measurement was performed by following previously reported procedures ,27,28,29q) were collected at the 4C SAXS II beamline at Pohang Light Source (PLS-II) in Korea. Monochromatic X-ray pulse with the energy of 16.90 keV was used for data collection. Static X-ray scattering patterns were recorded using a MarCCD detector . The sample-to-detector distance was set at 1 m. The q ranges of the data for dark and light states were 0.03–0.50 Å−1 and 0.03–0.43 Å−1, respectively. The protein was dissolved in the buffer solution of 20 mM Tris (pH 7.0) with 20 mM NaCl and the concentration of the protein was 10 mg/mL. The sample solution was centrifuged at 10,000 g for 10 min to remove unwanted aggregated particles and only the supernatant was used for the measurement prior to performing the SAXS measurements. To populate the light state, the protein sample was continuously irradiated by focused blue LED light during the measurement. To avoid any damages induced from X-ray and optical lights, the X-ray scattering curves of the SML-PYP were obtained by employing a flow cell system . One-dimensional scattering data, I(q), were obtained by azimuthal averaging. Scattering intensities for the pure buffer solution were measured by a separate experiment to determine the background intensity, and the I(q) were extrapolated to q = 0 by GNOM program. The Rg value and the pair distribution function, P(r), were calculated through indirect Fourier transformation [−1 and 0.00–50.06 Å, respectively, and those for the light state of SML-PYP are 0.05–0.40 Å−1 and 0.00–54.16 Å, respectively. The maximum dimension (Dmax), which is the maximum distance between scatterers was determined from PRIMUS at ATSAS. Using DAMMIF program [−1 and the optimal P(r) function was performed to reconstruct the shape of the protein. This shape reconstruction was repeated nineteen times for each data. Representative structures were generated by superimposing each structure determined from DAMMIF process result onto a template structure using DAMSEL and DAMSUP. Nineteen low-resolution dummy atomic models were averaged using DAMAVER [The SAXS data were processed and analyzed using the ATSAS package


With exposure to UV, transformation from trans-isomer to cis-isomer of HA-AZO could be realized according to UV spectra. Reversely, trans-isomer could be gradually recovered from cis-isomer in the dark. Simultaneously, quick response and slow recovery could be detected in the process of structural change. Moreover, repeated illumination was further used to detect the antifatigue property of HA-AZO, which showed no sign of fatigue during 20 circles. The influence of pH value on UV spectrum for HA-AZO was investigated in the work. Importantly, in acid solution, HA-AZO no longer showed any photoresponsive property. Additionally, the status of HA-AZO under the effect of UV light was investigated by DLS results and TEM image. Finally, in vitro cytotoxicity evaluations were performed to show the effects of photoresponsive macromolecule on cells.Azobenzene, a photo switcher, has attracted increasing interest due to its structural response to photo stimulus in the field of information science and chemical sensing in the recent decades. However, limited water solubility and cytotoxicity restrained their applications in the biomedical field. In research, HA-AZO has been designed as a water soluble photo switcher in biomedical application. Synthesized HA-AZO had good water-solubility and a stable π-π Photoresponsive molecules, which cause reversible changes, i.e., their chemical and physical properties changes due to structural change with a light stimulus, have attracted increasing interest in the field of information science and chemical sensing in the recent decades  and molecular aggregation in water was also confirmed by transmission electron microscope (TEM) images. Finally, cell viability and apoptosis profile was used to evaluate the cytotoxicity of photoresponsive macromolecule.Although azobenzenes have been intensively investigated, work still needs to be done to obtain a biocompatible photoresponsive macromolecule for biomedical application, characterize their structure and assess their properties including cytotoxicity properties.w = 1,000 kDa) was obtained from Shandong Furuida Co., China. Trypsin, Dulbecco's modified Eagle's medium (DMEM), fluorescein diacetate (FDA), and 3- thiazol-2,5-dimethyl tetrazolium bromide (MTT) were obtained from Sigma. Fetal bovine serum (FBS) was purchased from Sijiqing biotech. Co., China. All other reagents and solvents were of analytical grade and used as received.1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), 2-morpholinoethane sulfonic acid (MES), and p-aminoazobenzene (AZO) was purchased from Aladdin. Hyaluronate acid . Briefly, 500 mg of EDC and 200 mg of NHS were successively added to 10 mL of 2% HA solution with magnetic stirring. Meanwhile, 10 mg of AZO was dissolved in 10 mL DMF. After 30 min, 100 mg of MES was further added to HA solution. HA solution and AZO solution were subsequently mixed together and the reaction was maintained for 5 h at room temperature. Then, the final solution was dialyzed with a dialysis bag of 10 kDa cut-off molecular weight for 3 days to remove unreacted chemicals and byproduct of small molecular weight. Finally, HA-AZO was obtained by freeze drying at −60°C at a pressure of 7–8 Pa.1H nuclear magnetic resonance  using D2O as solvent. HA-AZO water solution, AZO water solution, and AZO DMSO solution was characterized by UV spectroscopy (Cary 50). UV lamp (10 W) was used as photosource to induce tran-to-cis transition of AZO domain. In order to track the structural change of molecules, real-time UV spectra as a function of irradiation time and recovery time was recorded. Repeated irradiation and recovery method were applied to demonstrate the fatigue resistance of molecules. Macromolecule status in water solution was characterized by dynamic light scattering  and transmission electron microscope .HA-AZO was characterized by 2 at 37°C. The used cells were detached using 0.25% trypsin in PBS for the experiment. Simultaneously, HA-AZO and AZO were dissolved in DMEM with certain concentration separately with the same molar ratio. Then, 100 μL of above-mentioned solution was added into each well of 96-well culture plate, and into which the 100 μL cell suspension containing 5,000 cells was subsequently added. Cells were characterized as a function of cultural time. MTT assay was used to characterize cytoviability. Briefly, 20 μL MTT was supplemented into wells of the culture plate, which were then put back to continually culture cells for another 4 h. Two hundred microliters DMSO was added to dissolve the formed formazan pigment. The absorbance of above solution at 490 nm was recorded by a microplate reader (Infinite M200 PRO).HUVEC cells were incubated in a humidified atmosphere of 95% air and 5% COBesides MTT assay, apoptosis profile of cells was characterized by PI/FITC double staining (Annexin V). Briefly, 1 mL of above-mentioned HA-AZO solution or AZO solution was added into each well of 12-well culture plate, into which 1 mL cell suspension containing 200,000 cells was subsequently added. Cells of each well were detached using 0.25% trypsin without EDTA after culturing for 24 h separately. Detached cells were washed with 1 × binding buffer, resuspended in 500 μL 1 × binding buffer, and subsequently ordinal stained by PI solution and FITC solution for 15 min. The stained cells detected by flow cytometry  within 1 h in dark.t-test for differences. Results were reported as means ± standard deviation. The significant level was set at p < 0.05.Data were analyzed using the 1H NMR as shown in Figure 3-O at 1 position, the chemical shifts from 3.1 to 4.0 ppm are attributed to the protons of pyranose ring, and the chemical shift at 7.9 ppm is attributed to the protons of benzene ring of AZO at 2 position. The chemical shift at 2 position confirmed successful grafting of AZO onto HA main chain. Besides qualitative analysis, 1H-NMR provided quantitative information since areas of resonance peaks are proportional to number of protons. According to areas in Figure 3-O, which is 2.2% (per two pyranose ring).Synthesized HA-AZO was characterized by * transition for trans-isomer decreased significantly and the peak at 420 nm belonging to n-π* transition for cis-isomer increased a little with irradiation time until 60 s, which was an obvious sign to indicate that the trans-form had been transferred to the cis-form  that coexists with trans-form structure in natural state, which indicates that trans-to-cis transition is induced in acid environment. However, no defined deduction has been verified by sufficient proof. Furthermore, since AZO molecule in water exhibited no trans-to-cis transition due to aggregation of molecules according to Figure Since pH value is also an important factor in physiological environment, UV spectra of HA-AZO solution with different pH value was tracked as shown in Figure On account of π-π stacking interaction between AZO domains, HA-AZO macromolecule might be in aggregation state, which was characterized by DLS and TEM as shown in Figure in vitro culture of HUVEC cells. In this method, cells were cocultured with HA-AZO on TCPs, and simultaneously, cytoviability and cell apoptosis profiles were used to evaluate the biocompatibility of HA-AZO. As shown in Figure Since biocompatibility of the macromolecule was an essential factor for its biomedical application, preliminarily cytotoxicity assessment was performed by In the cell apoptosis profile, live cells on TCPs without HA-AZO were approaching 78% and the dead cells were estimated to be 18% after cells were cultured for 3 days Figure . Further* transition absorbance peak trans-isomer. Upon UV for 5 min, transformation from trans-isomer to cis-isomer of HA-AZO could be realized according to UV spectra. Reversely, trans-isomer could be gradually recovered from cis-isomer in 1 h in dark. This characteristics revealed the reversible photo-responsive property of HA-AZO molecule with quick response and slow recovery. Moreover, antifatigue property of HA-AZO was confirmed by repeated illumination. The blue shift from 360 to 320 nm and emerging peak at 500 nm were found in low pH value solution. In acid solution, HA-AZO no longer showed any photo responsive property. HA-AZO was in small aggregation and its size was influenced by irradiation and varied with recovery time. Finally, in vitro evaluation showed little acute cytotoxicity, but the cell growth could be inhibited by HA-AZO. These properties indicated that HA-AZO was an optimal photo responsive macromolecule for biomedical application. The toxicity of AZO has been verified not only by our results but also by other researchers. Hence, their application in biomedical field is rarely reported. While the AZO grafting product HA-AZO exhibits little acute cytotoxicity from the result of both cell viability and cell apoptosis profile after 1 d, acute cytotoxicity is evaluated with 24 h before cell division. Theoretically, cell viability should multiply and increase with cultured days, since cells divide in every 24 h. Actually, cell growth viability showed little increase with cultured days. However, the increased rate is much lower than both that of TCPs control and theoretical calculation. These results showed that HA-AZO influenced cell proliferation and cell division, which indicated that the grafting product might have some genetic toxicity.HA-AZO was successfully synthesized with DS of 2.2%. Synthesized HA-AZO had good water-solubility and a stable π-πJP designed the macromolecule and predicted its property. ZG synthesized the macromolecule. LZ and HW investigated the properties of the macromolecule. XH was responsible for the whole manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Vibrio cholerae, both QS and the second messenger 3’, 5’—cyclic diguanylate (c-di-GMP) play a central role in controlling motility, motile-to-sessile life transition, and virulence. In this study, we found that water-soluble extract from the North American cranberry could significantly inhibit V. cholerae biofilm formation during the development/maturation stage by reducing the biofilm matrix production and secretion. The anti-biofilm effect by water-soluble cranberry extract was possibly through modulating the intracellular c-di-GMP level and was independent of QS and the QS master regulator HapR. Our results suggest an opportunity to explore more functional foods to fight stubborn infections through interference with the bacterial signaling systems.Quorum sensing (QS) and nucleotide-based second messengers are vital signaling systems that regulate bacterial physiology in response to changing environments. Disrupting bacterial signal transduction is a promising direction to combat infectious diseases, and QS and the second messengers are undoubtedly potential targets. In  Quorum sensing (QS) and the nucleotide-based second messengers, especially the cyclic dinucleotides, are two central signaling systems utilized by many bacteria to regulate their physiological functions in response to changing environmental conditions or during the developmental process. Due to their decisive roles in bacterial physiology, QS and the second messengers have been considered as potential targets for new drug development to tackle the increasingly grim situation of antibiotic resistance. By blocking the signaling transduction rather than targeting the essential genes, placing selective pressure on resistant strains of bacteria is avoided. In the past twenty years, natural QS inhibitors (QSIs) have been identified from a number of organisms, and a list of synthetic QSIs have also been developed in research labs , 2. In cVibrio cholerae, both QS and the cyclic dinucleotides play a fundamental role in controlling cell motility, biofilm formation, and virulence gene expression. V. cholerae is the causative agent of a frequently fatal disease called cholera. Since the first cholera pandemic occurred ~ 200 years ago, the disease has affected millions of people. With a better knowledge of the disease control and improved water and sanitation facilities, the disease transmission has been eliminated in the developed countries, yet cholera remains a threat in many parts of the developing world. An essential component to this pathogen’s success and persistence in the environment is its ability to attach to both biotic and abiotic surfaces via biofilm formation [V. cholerae enters the body, it must first survive the acidic environment of the stomach and then proceed to attach to the intestinal wall. Biofilms provide cells resistance to high acidity and therefore are critical for the transmission and infectivity of V. cholerae [V. cholerae biofilm, Vibrio polysaccharide, is synthesized by enzymes encoded in the two vps (Vibrio polysaccharide synthesis) gene operons (vps-I and vps-II) [rbmA, rbmB, and rbmC) as well as bap1, which is located downstream of vps-II, are involved in the production of matrix proteins and maintenance of the biofilm structure. RbmA provides cell-cell adhesion, RbmC and Bap1 form the envelopes to encase the cell clusters, and RbmB, whose function is yet to be characterized, is proposed to help with the detachment of bacteria from mature biofilms [vps operons are positively regulated by two major transcriptional regulators, VpsR and VpsT [rbmA, rbmB, rbmC, and bap1 are positively regulated by VpsR [vpsR and vpsT is regulated by the cell density through the upstream QS pathway, and by the intracellular concentration of 3’, 5’—cyclic diguanylate (c-di-GMP), an important second messenger identified in a wide variety of bacteria [In the Gram-negative bacterial pathogen ormation . Biofilmcholerae , 6. The  vps-II) , 8. Addibiofilms –11. The and VpsT , 13, and by VpsR . Expressbacteria –17.V. cholerae, genes involved in biofilm formation and virulence are maximally expressed at low cell density and are turned off at high cell density. This unique system is thought to aid in V. cholerae’s life cycle during acute infection, allowing it to colonize in the intestine and promoting infection at low cell density, and helping the bacteria detach and transition back to its natural environment at high cell density [V. cholerae responds to at least two QS signaling molecules , CAI-1 and AI-2, through the response regulator LuxO. At low cell density, LuxO is in the phosphorylated form and activates expression of a set of small regulatory RNAs, which in turn inhibits expression of the major QS regulator HapR, allowing expression of vpsR, vpsT, as well as virulence genes. At high cell density, LuxO is dephosphorylated; thus HapR is de-repressed and inhibits biofilm and virulence genes.Unlike many other pathogenic bacteria that cause persistent infections in which QS typically activates biofilm formation and virulence at high cell density, in  density , 18, 19.V. cholerae, increasing the intracellular c-di-GMP level promotes the transition from motile to sessile lifestyle [V. cholerae genome encodes 31 proteins with the GGDEF domain, 12 proteins with the EAL domain, nine proteins with the HD-GYP domain, and ten proteins with both GGDEF and EAL domains [V. cholerae c-di-GMP level and biofilm formation [In ifestyle –22. C-diifestyle . Both thifestyle , 25. C-difestyle . The V.  domains , 28. Priormation , 29, 30.water-soluble cranberry extract standardized to 4.0% proanthocyanidins (WCESP) to Caenorhabditis elegans, a nematode model, could significantly enhance the host’s resistance to infection by various bacterial pathogens including Vibrio cholerae. Mechanistic studies showed that the WCESP-mediated protection was mainly through promoting the host’s innate immunity. Under our experimental condition, WCESP treatment did not affect the growth of V. cholerae or expression of the major bacterial virulence genes, but a slight reduction of bacterial colonization within C. elegans intestine was noticed [V. cholerea biofilm formation and further investigated the inhibition mechanism. We found that low concentration of WCESP could significantly impede V. cholerae biofilm formation during the development/maturation stage; this inhibition is independent of the QS pathway but is possibly through modulating the intracellular c-di-GMP level. This study suggests that cranberry contains certain active constituents that can interfere with V. cholerae c-di-GMP signal transduction and provides a potential means to control V. cholerae biofilm.Previously, we reported that supplementation of  noticed . In this6) was obtained from Dr. Andrew Camilli (Tufts University) [V. cholerae strains (except the MO10 strain) used in this study were all derived from the wild-type C6706 strain (O1 serotype El Tor isolated from Peru) [hapR-lacZ fusion, luxO- (luxO deletion), hapR- (hapR deletion), luxOC (constitutively active LuxO), cqsA- (cqsA deletion), luxS- (luxS deletion), cqsA-/luxS- (cqsA and luxS double deletion) strains were obtained from Dr. Jay Zhu (University of Pennsylvania) [Staphylococcus aureus , Pseudomonas aeruginosa , Enterococcus faecalis , Salmonella typhimurium , E. coli O157:H7 (ATCC#700927), Listeria monocytogenes (L. monocytogenes) ScottA (serotype 4b), and L. monocytogenes Mac (serotype 1/2a). The E. coli, V. cholerae, P. aeruginosa, and S. typhimurium strains were cultured in Lysogeny broth (LB). The S. aureus, E. faecalis, and L. monocytogenes strains were cultured in brain-heart infusion (BHI) medium.The plasmid pBAD33 was obtained from Coli Genetic Stock center (CGSC), and pAT1662 (pBAD33::VCA0956-Hisversity) . The V. ylvania) , 34. Theylvania) , 36, alsThe cranberry extract used in this study was obtained from Naturex-DBS, LLC  as described in previously published studies . A stockV. cholerae, and 37°C for other bacterial strains. Quantifications of sessile/biofilm cells were conducted by the crystal violet staining method and the direct enumeration of live cells. For the staining method, the planktonic bacterial cells were carefully removed from each tube, and the tube was washed twice with PBS. The sessile bacterial cells were stained with 1% crystal violet for 30 minutes, followed by distilled water wash for five times. To enumerate biofilm bacterial cells, planktonic cells were carefully removed from underneath the biofilm pellicle. The pellicle and surface-attached cells were washed twice with PBS and resuspended in 1 ml of PBS. The cells were vortexed with glass beads (ϕ = 1mm) for two minutes to disrupt the biofilm and then plated onto LB agar plates with proper dilutions. Colonies were counted on the second day. In a parallel set of experiments, total bacterial cells (both planktonic and biofilm cells) were measured by first vortexing the cells with glass beads and then plating onto LB agar plates with proper dilutions. Data shown are the average results from three independent experiments, and triplicate samples were tested each time. The data were pooled and analyzed using unpaired Student’s t-test. A p-value < 0.05 was accepted as statistically significant.Overnight bacterial cultures were inoculated (1:100 dilution) into 1 ml of LB or BHI medium in the presence or absence of 2 mg/ml of WCESP and incubated statically in polystyrene tubes for 24 hours to allow biofilm development. The temperatures used to develop biofilms were 25°C for V. cholerae was inoculated into 1ml of LB in polystyrene test tubes (1:100 dilution) and incubated statically at 25°C for 4 hours. Then, 40 μl of WCESP stock solution (50 mg/ml) was added to the test tubes to reach a final concentration of 2 mg/ml. In the control tubes, 40 μl of sterile ddH2O was added. The tubes were incubated at 25°C under static condition for the remaining 20 hours. Biofilm cells were measured using the standard plate count method as described above. Data shown are the average results from three independent experiments, and triplicate samples were tested each time. The data were pooled and analyzed using unpaired Student’s t-test. A p-value < 0.05 was accepted as statistically significant.The overnight culture of V. cholerae were inoculated into 1 ml of LB with or without 2 mg/ml WCESP in polystyrene test tubes (1:100 dilution). Cultures were incubated with shaking at 150 rpm at 25°C until reaching mid-log phase. Three microliters of the culture were dropped onto motility assay plates (LB with 0.3% agar). The plates were incubated at 25°C. The diameters of the motility zones were measured after 4 and 24 hours, respectively.Overnight cultures of V. cholerae C6706 culture was inoculated (1:100 dilution) into LB with or without 2 mg/ml of WCESP and incubated at 25°C with gentle shaking. Cell samples were collected at 4 and 24 hours respectively and washed twice with 0.85% NaCl. Samples were then transferred to glass test tubes and resuspended in 3 ml of 0.85% NaCl. Spectrophotometer reading was then taken from each sample at 600nm and recorded as the OD Initial. Next, 0.25 ml of toluene was added to each sample and vortexed for 2 minutes. Samples were then allowed to equilibrate at room temperature for 30 minutes. Following the complete separation of the toluene phase from the aqueous phase, OD of the aqueous phase of each sample was measured at 600nm and recorded as the OD Final. The Hydrophobicity Index (HPBI) was then calculated using the following formula: HPBI =  / OD Initial x 100%. Data shown are the average results from three independent experiments, and triplicate samples were tested each time. The data were pooled and analyzed using unpaired Student’s t-test. A p-value < 0.05 was accepted as statistically significant.The cell-surface hydrophobicity was measured based on the method described by Prabu et al. . OvernigV. cholerae C6706/PhapR-lacZ was inoculated into LB medium (1:100 dilution) with or without 2 mg/ml WCESP. Cultures were incubated statically at 25°C, and 1 ml of planktonic bacterial sample was collected at 4, 8, 16, and 24 hours, respectively. The β-galactosidase activity of each sample was measured according to the method developed by Miller [p-value < 0.05 was accepted as statistically significant.The overnight culture of y Miller . Data shV. cholerae C6706 culture was inoculated (1:100 dilution) into 1 ml of LB with or without 2 mg/ml WCESP. Cultures were grown statically at 25°C. Planktonic bacterial samples were harvested after 4 and 24 hours of incubation and washed once with PBS. Total RNA was prepared using RNAzol RT reagent  and stored at -80°C. Complementary DNA was synthesized using the iScript cDNA synthesis kit (Bio-Rad). qRT-PCR was performed using SensiFAST SYBR No-Rox Kit (Bioline) and the CFX96 real-time PCR detection system according to the manufacturer’s suggested protocol (Bio-Rad). The qRT-PCR conditions were: 95°C for 2 minutes, followed by 40 cycles of 5 seconds at 95°C, 10 seconds at 50°C, and 15 seconds at 72°C. 16S rRNA was used as the endogenous control to normalize the expression levels of target transcripts [−ΔΔCT) method [p-value < 0.05 was accepted as statistically significant. The primers used in this study are listed in Overnight nscripts . Relativ) method . Cycle tV. cholerae C6706 culture was inoculated (1:100 dilution) into LB medium and incubated statically at 25°C. The planktonic cells from the static cultures were collected at 4h, 8h, 16h, and 24h time-points to represent the key growth phases, and the total cell number in each sample was determined using the plate count method. Bacterial cells were pelleted by centrifugation and resuspended in 1 ml of 10 mM Tris-HCl (pH 8.0) buffer containing 100 mM NaCl. The cells were lysed by sonication. Trichloroacetic acid was added to the cell lysate to a final concentration of 12% to precipitate cellular macromolecules. The suspension was incubated on ice for 10 minutes and neutralized by 3 M KOH containing 0.4 M Tris and 2 M KCl, followed by centrifugation at full speed for 10 minutes. The supernatant was filtered through a 0.2 μm filter and then 1:10 diluted into the reaction buffer (10 mM Tris-HCl (pH 8.0) containing 1M NaCl), incubated at 95°C for 5 minutes, and cooled to room temp. TO was then added to each sample at a working concentration of 30 μM and incubated at 4°C for 12h until fluorescence readings were taken at excitation and emission wavelengths at 508nm and 557nm, respectively. The concentration of c-di-GMP was calculated using a calibration curve and normalized to 1 x 109 bacterial cells in 1 ml.The thiazole orange (TO)-based fluorescent detection method was used to quantify the intracellular c-di-GMP level . In brieV. cholerae biofilm formation, we grew V. cholerae wild-type C6706 strain statically in LB medium at 25°C, a temperature that more closely resembles the natural condition. WCESP was added into the growth medium at the same time during inoculation to reach a final concentration of 2 mg/ml. After incubation for 24 hours, the outcome of adding WCESP was palpable. As shown in V. cholerea is a human pathogen, we also tested WCESP’s ability to inhibit biofilm at 37°C. A similar inhibition effect was observed as well , pomB (encoding the flagellar motor protein), and mshA (encoding the type IV pilus) in the presence or absence of WCESP using qRT-PCR. No noticeable changes were detected after either 4 hours  are crucial for the initial attachment to abiotic surfaces, and the flagellum also mediates the spread across the surface area . Mass pr 4 hours  or 24 ho 4 hours . These r 4 hours . The samV. cholerae, the biofilm matrix protein Bap1 is the crucial determinant of pellicle mechanical strength and hydrophobicity, allowing the pellicle to spread and remain at the air-liquid interface [V. cholerae C6706 with and without 2 mg/ml WCESP and collected bacterial samples after 4 and 24 hours for the CSH measurement. As shown in Bacterial cell-surface hydrophobicity (CSH) depicts the tendency of a bacterial cell to aggregate with cells of similar hydrophobicity as opposed to water, which is mainly affected by the bacterial cell surface composition and structure . CSH alsnterface . To testvpsD (the fifth gene of the vps-I operon), vpsL (the first gene of the vps-II operon), as well as genes encoding the matrix proteins bap1, rbmA, rbmB, and rbmC, was evident in the presence of WCESP . The extracellular protein secretion (eps) system plays a vital role in V. cholerae pathogenesis and environmental survival; it is involved in exporting toxins and VPS across the outer membrane [epsE (the third gene of the eps operon) was measured to denote the change of the eps operon when exposed to WCESP. As shown in Using qRT-PCR, the expression of genes that are responsible for the production of VPS and matrix proteins was compared in the presence and absence of WCESP at 4 and 24 hours. No significant difference was detected at 4 hr. At 24 hr, downregulation of the of WCESP . Expressmembrane . ExpressV. cholerae biofilm, and cranberry constituents have been reported to attenuate QS-mediated cell-cell signaling in V. harveyi and P. aeruginosa [V. cholerae biofilm to the effects on QS system. In V. cholerae, high cell density (when CAI-1 and AI-2 concentrations are high) results in downregulation of biofilm formation through AI-sensing, LuxO dephosphorylation, and activation the master QS regulator HapR expression. HapR then inhibits vpsR, vpsT, and the vps-I, vps-II, and rbm genes. We reasoned that if the inhibition is through QS, then (1) WCESP would enhance the expression of HapR, and (2) in QS-deficient mutants , WCESP would no longer inhibit biofilm formation. To test this, we first examined the hapR transcription using a strain carrying a chromosomal hapR-lacZ fusion [hapR transcription was slightly lower (less than 2-fold) in the presence of WCESP at T = 4, 8, and 16 hr, but at T = 24 hr, hapR transcription was marginally higher (1.3-fold). qRT-PCR of hapR from 24 hr WCESP-treated bacterial culture showed no distinct change , cqsA- (no CAI-1), luxS-(no AI-2), and a cqsA-/ luxS- double deletion. The luxO- mutant was included to serve as a biofilm-negative control, as HapR is overexpressed in luxO- and strongly inhibits biofilm formation. The results are shown in QS pathway plays a central role in the regulation of ruginosa –48. ThusZ fusion . As showt change . ResultsV. cholerae that regulates the planktonic to biofilm transition and the biofilm matrix production. To examine WCESP’s influence on c-di-GMP, we sought to directly measure the intracellular c-di-GMP concentration using the thiazole orange (TO)-based fluorescent detection method [V. cholerae C6706 was grown statically at 25°C in the presence and absence of 2 mg/ml WCESP. The planktonic portion of the static cultures was collected at 4, 8, 16, and 24 hours, respectively. As the bacterial cell number changes during the growth phase and also is affected by the presence of WCESP, we normalized the final c-di-GMP concentration to 1 x 109 bacterial cells in 1 ml. As shown in V. cholerae biofilm formation by WCESP is possibly through reducing the intracellular c-di-GMP level during the development/maturation stage of the biofilm formation. If this holds true, then increasing the c-di-GMP level would at least partially counteract WCESP’s anti-biofilm effect. To test this, pAT1662 (a plasmid that contains the diguanylate cyclase VCA0956 gene under the control of the arabinose-inducible araBAD promoter) [V. cholerae C6706 by electroporation. Biofilm formation of the resulting strain was examined when 0.2% of L-arabinose and 2 mg/ml of WCESP were added to the growth medium simultaneously. As shown in The bacterial second messenger 3’, 5’—cyclic diguanylate (c-di-GMP) is another vital signaling system in n method . To do tromoter)  was intrVaccinium macrocarpon) are rich in polyphenols, including anthocyanins, proanthocyanidins (PACs), flavonoids, and phenolic acid derivatives [Staphylococcus epidermidis biofilm formed on soft contact lenses [Pseudomonas aeruginosa biofilm [V. cholerae biofilm formation. Our results indicated that sub-lethal level of WCESP could inhibit V. cholerae biofilm formation during the development/maturation stage by reducing the production and excretion of VPS and biofilm matrix proteins. We further investigated the involvement of the two key signaling systems and found that the inhibition is independent of QS and the QS master regulator HapR. On the other hand, the intracellular c-di-GMP level was decreased in the presence of WCESP. Our results imply WCESP’s anti-biofilm effect is possibly through modulating c-di-GMP signaling, though we could not rule out the possibility that other regulatory pathways might be involved.Due to the selective pressure of antibiotics, bacterial resistance to antibiotics is becoming an increasing health threat worldwide. A growing body of evidence demonstrated that plant extracts offer considerable antimicrobial and anti-biofilm potentials and do not come with the significant risk of antibiotic resistance. A vast number of phytochemicals have been recognized as a valuable alternative and complementary medicine to treat bacterial infections , 50. Theivatives –53. The ivatives –56, dentivatives –60, as wivatives . There aivatives , 62–65 oivatives –68. Crant lenses , and Pse biofilm . DespiteV. cholerae, this is opposite; inhibiting QS (like the hapR deletion or the luxOC strain) would enhance biofilm formation. Initially, we hypothesized that cranberry extract might promote QS signaling, lead to the activation of HapR, which inhibits biofilm, but our results didn’t support this idea. Meanwhile, cranberry constituents have been reported to attenuate QS-mediated cell-cell signaling in V. harveyi and P. aeruginosa. In V. harveyi, the nondialyzable material (NDM) of cranberry competes with the autoinducer for binding to its receptor [P. aeruginosa, cranberry-derived PACs (contains > 95% PACs) inhibits autoinducer production and also antagonizes the activation of QS transcriptional regulators [In most bacterial pathogens, biofilm formation is activated at high cell density. Thus, inhibition of QS would result in the reduction of biofilm. But in receptor . In P. agulators . These sV. cholerae, c-di-GMP controls motile to sessile transition at both the initial attachment and the matrix production/development stages. Jones et al. reported that c-di-GMP promotes the polymerization of MshA subunits to form the MshA pili which are indispensable for the initial attachment to abiotic surfaces [V. cholerae genome encodes numerous proteins containing the functional domains for c-di-GMP metabolism, instead of measuring the expression of individual genes, we directly measured the change of intracellular c-di-GMP concentration when WCESP is added to the growth medium. The decrease of c-di-GMP was detected in the 8, 16, and 24 hr samples, but not in the 4 hr samples. Consistent with the results from the motility, qRT-PCR, and pre-established biofilm inhibition assays, this result suggested that the soluble constituents from WCESP impede the development/maturation stage of biofilm formation through modulating the c-di-GMP level. It’s worth mentioning that we didn’t observe enhanced motility at 24 hr, when c-di-GMP level was reduced by WCESP. High concentration of c-di-GMP is known to repress V. cholerea motility [The second messenger c-di-GMP has been identified in almost all major bacterial phyla, and its role as a signaling molecule involved in a variety of physiological processes has been well documented . In V. csurfaces . C-di-GMsurfaces , 25. Sinmotility , thus weS. aureus, E. faecalis, and two L. monocytogenes strains) and three Gram-negative bacteria . No inhibition was observed in all these strains. In contrast to V. cholerae, QS (high cell density) promotes biofilm formation in these strains. Because the content of PACs or NDM  is quite low in our WCESP sample, there was probably no impact on QS signaling in these strains. In the three Gram-negative bacteria, c-di-GMP signaling has also been characterized to regulate biofilm formation, yet we didn’t see biofilm inhibition effect. Maybe higher concentrations of WCESP are required, or more importantly, each bacterium has a unique c-di-GMP signaling network.In this study, we also tested WCESP’s anti-biofilm activity on other pathogenic bacteria, including four Gram-positive strains Click here for additional data file.S2 FigResults are the average of three independent experiments, and error bars are standard error of the mean.(TIF)Click here for additional data file.S1 TableThe same experiment was repeated three times. The data shown are the representative results from one of the experiments.(DOCX)Click here for additional data file.


Non-obstructive azoospermia is mostly irreversible. Efforts to cure this type of infertility have led to the applicationof stem cells in the reproduction field. In the present study, testicular cell-mediated differentiation of male germ-like cells frombone marrow-derived mesenchymal stem cells (BM-MSCs) in an Dazl, Piwil2 and Stra8 were evaluated by real-time polymerase chain reaction (PCR). In this experimental study, mouse BM-MSCs were isolated and cultured up to passage three.Identification of the cells was evaluated using specific surface markers by flow-cytometry technique. Four experimental groupswere investigated: control, treatment with retinoic acid (RA), indirect co-culture with testicular cells, and combination of RAand indirect co-culture with testicular cells. Finally, following differentiation, the quantitative expression of germ cell-specificmarkers including Dazl expression in the indirect co-culture with testicularcells group in comparison to the control group. Quantitative expression level of Piwil2 was not significantly changed incomparison to the control group. Stra8 expression was significantly higher in RA group in comparison to other groups. Molecular analysis revealed a significant increase in Dazl, in the induced cells. Combination of co-culture with testicular cells and RA did not show any positive effect on thespecific gene expressions.Indirect co-culture of BM-MSCs in the presence of testicular cells leads to expression of male germ cell-specificgene,  Azoospermia has always been the most challenging issue associated with male infertility treatment . The coEfforts to treat the non-obstructive azoospermia have led to the application of stem cells in this field. Researchers have applied various sources of stem cells such as embryonic stem cells , inducedBone marrow stromal/mesenchymal stem/precursor cells (BM-MSCs) are considered as multipotent stem cells which have the potential of self-renewal and differentiation to different types of cell, such as osteocytes, chondrocytes and adipocytes . MSCs arDazl), Piwi like homolog 2 (Piwil2) and stimulated by RA gene 8 (Stra8) are three specific male germ cell genes expressed at different stages of male germ cells production  gene family which encodes RNA binding proteins necessary for germ cell development in different organisms  are the founder population of male germ cells originated from proximal epiblast and migrate through the dorsal mesentery to rich the developing gonads . These coduction , 8, 10. rganisms . Piwil2,em cells . Stra8  meiosis . Stra8 rm cells .During spermatogenesis, different testicular cells -including germ, Sertoli, Leydig and peritubular myoid cells-interact with each other . TherefoDazl, Stra8 and Piwil2 expressions in BM-MSCs. Our general purpose was preparing a condition in which male germ-cell specific genes can significantly be expressed in BM-MSCs.In an indirect co-culture system, an insert filter with a biological microporous membrane is used which physically separates the upper compartment from the lower one, whereas it permits transfer of soluble factors through it . In thisIn this experimental study, Male Naval Medical Research Institute (NMRI) mice were housed under environmentally controlled conditions in 23-25°C and a 12/12 hours light/dark cycle. They were fed with a standard laboratory diet and accessed to drinking water ad libitum. Animals were treated in accordance with the Ethics Committee of Zanjan University of Medical Sciences . 2 culture flask  without any additional manipulation. The flushing was done several times, so that the lumen became pale. This method of collection of BM-MSCs is in accordance with Huang et al.  and passaged up to three times (P3). To identify BM-MSCs, surface antigens of the cells were evaluated by flow-cytometer. Concisely, cells at passage three were harvested and cell suspension was stained with fluorescence conjugated antibodies phycoerythrin-conjugated rat anti-mouse CD73, fluorescein isothiocyanate-conjugated rat anti-mouse CD44, phycoerythrin-conjugated rat anti-mouse CD90, fluorescein isothiocyanate-conjugated rat anti-mouse CD45 and phycoerythrin-conjugated rat anti-mouse CD34  for 45 minutes at 4°C. Following the wash with PBS, staining buffer was used and cells were ready for flow-cytometry analysis. Cells were incubated by isotype control anti-bodies to measure nonspecific background signals. Flow-cytometry analysis was performed by BD FACsort device .Male NMRI mice of 4-6 weeks were sacrificed by cervical dislocation. Animals were soaked in povidone-iodine for 2-3 minutes, then two tiny incisions were made at the skin and superficial fascia of lower limbs. The lower limbs were removed with a pair of scissors separating it from the hip joint and put on a sterile gauze. The accompanied soft tissue  was removed, and femurs and tibiae were separated and put in a dish containing phosphate buffered saline  and penicillin/streptomycin . The dish was transferred under a laminar hood. The bones were subsequently washed again with PBS and put on a sterile gauze to dry. Both ends of the bones were cut, then with an insulin syringe containing high glucose Dulbecco’s Modified Eagle Medium and 1% penicillin/streptomycin, all the contents of the bone’s lumen were flushed directly to 25 cmg et al. . At firTwelve male NMRI mice neonates (1-3 days old) were sacrificed. Mice were soaked in povidone-iodine for 2 minutes. Then a tiny cut through the skin, muscles and peritoneum were made at the lower part of the abdomen. By gently pressing the abdominal walls, intestinal loops, urinary bladder and accompanying testes were detectable. Intestinal loops and urinary bladder were set aside, so that we could see the tiny testes more clearly. By using a pair of tiny, sharp tip, sterile scissors testes were removed and put in a dish containing PBS and penicillin/streptomycin. The accompanying tissues of testis were removed and washed again with PBS. Testes were detunicated and smashed for enzymatic digestion in a 15 ml falcon . Five milliliter of trypsin-EDTA was added to the falcon and vigorously shaken. It was subsequently left at room temperature for 3 minutes. After additional vigorous pipetting, the relatively homogenous solution was centrifuged at 300 g for 5 minutes. Then, the supernatant was removed and FBS was added to a total amount of 12 ml. After pipetting, testicular cell suspension was ready for co-culturing. This method of preparing testis tissue is a modified approach of Lacham-Kaplan et al.  study . 5 cells/ well) in a 24-well plate  before preparing the testicular cells suspension. When BM-MSCs were reached to 70% confluence (0.05×105 cells/ well), the co-culture process was started. The 0.4 µm pore diameter insert filters  were located above the plates and then 0.5 ml of testicular cells suspension were applied over the filter (0.3×105 cells/filter). Culture medium (DMEM plus 1% FBS) was added to a proper amount, so that both compartments could interact with each other. Every day for seven days, 200 µl of the medium was removed and fresh 200 µl medium was added. After 7 days of co-culture, BM-MSCs were removed from the plates by trypsin-EDTA for RNA extraction and real-time polymerase chain reaction (PCR) analysis.BM-MSCs of passage three were plated (0.01×102 flasks with 80%confluence of BM-MSCs in passage 3 were cultured in DMEM plus 1% FBS); RA ; co-culture ; co-culture plus RA . BM-MSCs at passage three were investigated in 4 groups: Control , 1 µg purified RNA from cultured cells was used to synthesize 20 µl cDNA, according to the manufacturer’s instructions. cDNA was used to quantify was used . The seqData were analyzed by SPSS 16 software . All data are presented as means ± standard error of mean from 3 independent experiments. To compare differences of means in multiple tests, One-Way analysis of variance  was used. Relative quantification method was applied for real-time PCR analysis. Values of P=0.05 were considered statistically significant. The results showed that BM-MSCs were attached to the dish surface, after 24 hours cultivation. After adhering, BM-MSCs were fibroblast-like shape . By repBM-MSCs of passage three were analyzed for specific mesenchymal and hematopoietic markers using flow cytometry assay. Flow-cytometry results demonstrated that BM-MSCs of passage three are positive for CD73 (85.86%), CD90 (87.48%) and CD44 (78.12%), while it was negative for CD45 (0.8%) and CD34 2%, .Dazl, Piwil2 and Stra8 in the different experimental groups were examined using quantitative reverse-transcription real-time PCR. The results are presented relative to control group (BM-MSCs cultured in DMEM plus 1% FBS) (The changes in expression of  1% FBS) .Dazl in the study groups was significantly increased in comparison to the control group. Dazl mRNA expressions in the co-culture group (4.16 ± 0.28) was significantly up-regulated when compared to RA  and co-culture plus RA induction . Furthermore, Dazl expression was significantly higher in RAgroup compared to co-culture plus RA group , but it shows higher expression levels in co-culture (0.22 ± 0.03) and co-culture plus RA (0.22 ± 0.02) groups compared to RA group  was significantly up-regulated in comparison with co-culture  and co-culture plus RA group (P=0.001). No expression of Stra8 was detected in the co-culture plus RA group (The mean value of fold-change for RA group . In order to differentiate BM-MSCs toward germ-like cells, in this study, testicular cells suspension was used as an effective microenvironment in an indirect co-culture system. For better induction of differentiation, synergic effect of RA and testicular cells was also investigated.Dazl. Utilizing RA could increase Stra8 gene expression, considerably. Combination of RA and testicular cells did not show a positive effect on specific male germ cells genes expression.The results of this study indicated that indirect co-culture of BM-MSCs with testicular cells increased male germ cell-specific gene expression, BM-MSCs are commonly used in experimental studies, in terms of easy availability, isolation and culture. In addition, they are immunosuppressive and can be obtained from an adult person without ethical issues , 13. In in vivo condition (The relation of cell culture environments to what really happens in an organism can be enhanced by creating microenvironments behaving similar to ndition . Therefondition , stem cendition , leukemindition  and insundition , for BM-ndition . Sertolindition . Sertolindition . Since andition . SALL4A. This leads to higher expression of receptor tyrosine kinase(RA is small, polar molecule which easily passes thetissues and induces its action by binding to retinoidreceptors on nuclei . Bindinge kinase. Studiese kinase, 10, 30.e kinase, 31. Dazl is an important gene in differentiation and development and it may function as a master gene in germ cell differentiation (Dazl, Boule and Stra8. In the present study, Dazl expression is significantly increased by RA induction and indirect co-culture with testicular cells in comparison to the control group. Combination of RA and indirect co-culture with testicular cells did not lead to significant results in Dazl expression. Geens et al. (Dazl by the RA induction has been reported in other studies (Dazl in embryonic stem cells treated with RA, its expression is initially low and by time it is increased. We might have observed a higher quantitativegene expression if we continued the process of treatment and indirect co-culture. Piwil2 expression level did not show any significant change after 7 days of RA induction and indirect co-culture. It seems that temporal expression of Piwil2 is in a way which is decreased after 7 days of RA induction, it is evident in the study of Silva et al. (Piwil2, however higher expression rates were evident when they continued the treatment. Higher expression levels of Piwil2 are evident in days of 2 and 4 of RA induction, as well. Probable higher Piwil2 expression can be expected if we continue RA treatment for more than 7 days (Piwil2 as a mitotic gene, there are other studies reporting higher expression of this gene in spermatids and spermiogenesis (Piwil2 gene (Piwil2 expression is low in this study. Since Stra8 is the target gene of RA (Stra8 expression when stem cells are treated with RA (Stra8 expression.ntiation . In a rentiation , differs et al.  also coustudies , 31. Silstudies  has repoa et al. . In then 7 days , 10. Alogenesis  and indil2 gene . Therefo with RA , 9. SimuStra8 (Stra8 expression, as we can see in our study. The absorption of RA by testicular cells and theinhibitory effect of CYP26B1 are probable reasons whythe combination of RA and testicular cells could not leadto better differentiation of BM-MSCs. The combination of RA and testicular cells could not leadto better differentiation of BM-MSCs. It is hypothesizedthat RA is mostly absorbed by the spermatogonial cellsof the testis, bearing RA receptors . CytochrStra8 . So, theAlthough differentiation of male germ cells occurs in direct contact with Sertoli cells , endocriThe novelty of this study is related to indirect co-culture of testicular cells with BM-MSCs in the presence of RA. Our hypothesis was that different testicular cells and theirinteractions promote the Sertoli cells function and theymay serve as a proper microenvironment for induction of differentiation in BM-MSCs toward male germ like cells. Dazl. RA is effective to increase the gene expression, but combined effect of co-culture and RA does not increase the specific male germ cells genes expression.Indirect co-culture of testicular cells with BM-MSCs leads to significant increase in male germ cell specific gene


Middle ear  administration is a promising therapeutic method as it offers the possibility of achieving high inner ear drug concentrations with low systemic levels, thus minimizing the risk of systemic side effects and drug-drug interactions. Premature elimination through the Eustachian tube may be reduced by stabilizing drug solutions with a hydrogel, but this raises the secondary issue of conductive hearing loss.in vivo model of ototoxicity.This study aimed to investigate the properties of a chitosan-based particulate hydrogel formulation when used as a drug carrier for IT administration in an n = 94). Both contained the hearing protecting drug candidate sodium thiosulfate with different concentrations of chitosan gel particles (25% vs. 40%). The safety of the two systems was explored in vivo. The most promising system was then tested in guinea pigs subjected to a single intravenous injection with the anticancer drug cisplatin (8 mg/kg b.w.), which has ototoxic side effects. Hearing status was evaluated with acoustically evoked frequency-specific auditory brainstem response (ABR) and hair cell counting. Finally, in vivo magnetic resonance imaging was used to study the distribution and elimination of the chitosan-based system from the middle ear cavity in comparison to a hyaluronan-based system.Two particulate chitosan-based IT delivery systems, Thio-25 and Thio-40, were investigated in albino guinea pigs (p < 0.05) that remained after 10 days (p < 0.05) without evidence of hair cell loss, although the elevation induced by Thio-25 was significantly lower than for Thio-40 (p < 0.05). Thio-25 significantly reduced cisplatin-induced ABR threshold elevations (p < 0.05) and outer hair cell loss (p < 0.05). IT injection of the chitosan- and hyaluronan-based systems filled up most of the middle ear space. There were no significant differences between the systems in terms of distribution and elimination.Both chitosan-based IT delivery systems caused ABR threshold elevations (Particulate chitosan is a promising drug carrier for IT administration. Future studies should assess whether the physical properties of this technique allow for a smaller injection volume that would reduce conductive hearing loss. Cisplatin is a first-generation, platinum-based chemotherapeutic agent widely used in solid tumor treatment. However, cisplatin may cause permanent hearing loss and tinnitus, limiting its therapeutic use. There is no established pharmacological method to prevent or treat cisplatin-induced ototoxicity.At the cellular level, cisplatin-induced ototoxic effects are often manifested as loss of the cochlear outer hair cells (OHCs), as shown in experimental animals . ExcessiThe round and oval windows are the main routes of drug transport from the tympanic cavity to the inner ear . Some drin vivo model of ototoxicity.IT administration induces temporary conductive hearing loss, a factor which must be accepted by patients. Furthermore, ideally, a drug carrier should degrade in a controlled manner without inducing any side effects in the middle and inner ears. Here, we assessed the protective effects of a chitosan-based delivery system for sodium thiosulfate to inhibit cisplatin-induced ototoxicity over a 10-day period. The carrier was a homogenously deacetylated chitosan with physical and immunological properties different to those of traditional, heterogeneously deacetylated chitosans. The specific aim of the present study was to investigate a chitosan formulation, based on a dispersed particulate hydrogel in an aqueous suspension, as a drug carrier for IT administration in an n = 15) or Thio-40 (n = 15). Both groups received a single unilateral IT injection of sodium thiosulfate (100 mM) in a chitosan-based hydrogel formulation. The contralateral ear served as a control (no IT injection). ABR was measured prior to and 7 and 10 days after IT injection. Neither formulation caused hair cell loss, and ABR threshold shifts in the Thio-25 group were milder than in the Thio-40 group. Thio-25 was therefore used in the second part of the study.The study consisted of three parts. The first part investigated the safety of a chitosan-based delivery system for IT administration by exploring its effects on auditory brainstem responses (ABRs) to air-conducted pure tones and on hair cells. Two different candidate gel formulations were used, herein referred to as Thio-25 and Thio-40. These formulations differed in terms of the concentration of chitosan particles: 25% in Thio-25 and 40% in Thio-40. Guinea pigs were randomly assigned to one of two treatment groups: Thio-25 (n = 15), Placebo-25-cispt (n = 15), or NaCl-cispt (n = 15). All three groups received a single intravenous (i.v.) injection of cisplatin [8 mg/kg body weight (b.w.)]. One hour earlier, the Thio-25-cispt group was given a single unilateral IT injection of Thio-25, while the Placebo-25-cispt group received the vehicle only (Placebo-25), i.e., the same gel formulation (25% chitosan gel particles) but without sodium thiosulfate. The NaCl-cispt group was given an IT injection of sodium chloride . The contralateral ears were not injected in any group. Auditory function was evaluated by measuring ABR prior to injection and then 10 days later.The second part investigated the protective effects of Thio-25 IT administration on cisplatin-induced ototoxicity. Guinea pigs were randomly assigned to three treatment groups: Thio-25-cispt  received single IT injections of a paramagnetic gel based on chitosan (Chito-Dota) and hyaluronan (Hya-Dota). All animals received Chito-Dota in one ear and Hya-Dota in the contralateral ear. The gels were then visualized in vivo using magnetic resonance imaging (MRI), which was carried out on three occasions per animal over a 2-week period after gel injection. The animals were euthanized immediately after the final MRI session.The third part investigated the behavior of a chitosan-based formulation in the tympanic cavity compared with a hyaluronan-based formulation. Guinea pigs  subcutaneously  each day after cisplatin exposure. All animal procedures were performed under anesthesia and aseptic conditions and were in accordance with the Swedish national regulations for animal care and use. The experimental protocol was approved by the Regional Ethical Review Board in Uppsala (No. C5/15) and the Regional Ethical Review Board in Stockholm (No. 138/15).Our experiments involved 94 guinea pigs . Both sexes were utilized, and b.w. ranged from 263 to 413 g. Animals were maintained in an enriched environment in small groups on a 12-h light/12-h dark cycle at a temperature of 21°C and 60% humidity. They were given access to water and standard chow In parts one and two of the study, general anesthesia was produced with ketamine  and xylazine . Anesthesia depth was assessed by measuring the pedal reflex, and additional doses of ketamine (25 mg/kg b.w.) were given if needed. Bupivacaine  was used for local anesthesia. Buprenorphine  was used as a post-treatment analgesia in animals subjected to cisplatin administration.In part three of the study, general anesthesia was induced in an induction box using 4–5% isoflurane in a 3:7 oxygen:air gas mixture after pretreatment with atropine  and 100% oxygen for 30 min to reduce respiratory tract secretions. The animal was then quickly transferred to an MRI-compatible rig where anesthesia was maintained using 2–3% isoflurane. Lidocaine  was used for local anesthesia. Anesthesia depth was monitored by measuring the pedal reflex and visual inspection of the respiratory rate. Under MRI, anesthesia depth was monitored by automatic assessment of heart and breathing rates.N-deacetylation 49%, viscosity 365 mPas; ViscosanTM, Flexichem AB, Uttran, Sweden) was suspended in 185 mL of distilled water  and dissolved in 2 M aqueous HCl, which was added dropwise. The pH of the solution was adjusted to neutral with 1 M aqueous NaOH. The total volume was then adjusted to 200 mL with distilled water. The solution was filtered through a 5-μm syringe filter and heat-sterilized at 121°C for 20 min. Then, 12 g of this solution was mixed with a solution of sodium thiosulfate  and another 7.2 g of distilled water. The resultant solution was then filtered through a 5 μm syringe filter. 3,4-Diethoxy-3-cyclobutene-1,2-dione  was then added to 22.8 mL of the chitosan solution, and the mixture was stirred for 15 min at room temperature. The solution was then placed in a heating cabinet at 40°C for 14 days, after which the solidified gel was mechanically processed into 10-μm particles. Then, 1.29 g of gel particles were added to 3.87 g of a 30 mM phosphate buffer containing 100 mM of sodium thiosulfate to yield a 25% (w/w) ViscoGelTM suspension. Thio-40 was prepared using the same method but by mixing 2.08 g particles with 3.12 g of the phosphate/thiosulfate buffer. A placebo gel to Thio-25, referred to as Placebo-25, was prepared in a similar way as Thio-25 but without the addition of sodium thiosulfate.To prepare Thio-25, chitosan  was suspended in 90 mL of distilled water. Then, 530 μL of 2 M aqueous HCl was added, and the chitosan concentration adjusted to 0.5% with water. DOTA-NHS-ester  was dissolved in 20.0 mL of distilled water. Then, 14.0 mL of this solution was added to 23.51 mL of the chitosan solution. The pH was adjusted to 7.02 with 1 M NaOH, and the mixture stirred for 2 days. Gadolinium (III) acetate hydrate  was dissolved in distilled water (20.0 mL); 14.6 mL of this solution was then added to the chitosan-DOTA solution, and the mixture was stirred overnight. Then, 270 mg of EDTA  was added, and the mixture stirred for 30 min. The solution was dialyzed against 3 × 3 L distilled water . Subsequently, the dialysate was lyophilized; 86.8 mg of the lyophilized powder was dissolved in distilled water, the pH adjusted to 6.7, and the volume adjusted to 6.5 mL. To 3.75 g of this solution, diethoxy-3-cyclobutene-1,2-dione  was added while stirring. After 15 min of stirring at room temperature, the solution was placed in a heating cabinet at 40°C for 14 days. Finally, the solidified gel was mechanically processed into 10-μm particles, and 2.068 g of gel particles were mixed with 6.2 mL of 30 mM phosphate buffer.Chitosan  was then added under vigorous stirring to 209 mg of sodium hyaluronate . Then, N-dimethylaminopropyl-N ethyl carbodiimide hydrochloride  was added under continuous stirring. This solution was slowly added to the ethylene diamine solution under vigorous stirring, and the reaction mixture was left at room temperature for 4 h. The solution was then dialyzed against 4 × 5 L distilled water , after which the dialysate was lyophilized. Then, 138 mg of the lyophilized powder was dissolved in distilled water; 34.1 mg of DOTA-NHS-ester was added, the pH was adjusted to 6.9, and the mixture was stirred overnight. Then, 41 mg of gadolinium (III) acetate hydrate in 4 mL distilled water was added, and the mixture was stirred overnight. Finally, 270 mg of EDTA was added, and the mixture was stirred for 30 min followed by dialysis against 3 × 4 L of distilled water . The dialysate was subsequently lyophilized, and 54.4 mg of the lyophilized powder was dissolved in 5.44 mL of 30 mM phosphate buffer.Ethylene diamine  was dissolved in 20 mL of distilled water, pH was adjusted to 6.0 with 2 M HCl, and the volume was adjusted to 25.0 mL. All animals were subjected to a single gel injection (approximate volume: 0.15 mL) into the auditory bulla following a small paracentesis of the tympanic membrane.Cisplatin  was injected at a rate of 0.2 mL/min through a catheter  inserted into the right jugular vein toward the heart. To rinse the catheter, 1 mL of sterile saline was administered immediately after cisplatin injection. The catheter was subsequently removed, the jugular vein was ligated, and the skin was sutured.Auditory function was quantified by determining the hearing thresholds at 12.5, 20.0, and 30.0 kHz with air-conducted acoustically evoked ABR. Each animal was placed in a soundproof box. The frequency-specific stimulus signal was generated through a signal analyzer  controlled by a computer and presented through an electrostatic speaker . The speaker was connected to a 10-cm tube positioned in the ear canal of each guinea pig. Neural responses were then collected using three subdermal electrodes: one placed at the vertex (active), one on the mastoid (reference), and a ground electrode on the lower back. The ABR threshold was defined as the lowest stimulus intensity that produced a reproducible response for ABR wave II, which was visualized at the same latency after an average of 1,000 recordings.After the final ABR assessment, animals were deeply anesthetized with sodium pentobarbital  and subsequently decapitated. In the Thio-25, Thio-40, and Thio-25-cispt groups, the temporal bones were removed and the bulla opened to expose the cochleae. Small fenestrations were made in the apex and round window, and 4% phosphate-buffered formaldehyde was gently flushed through the cochlea. Surface preparation was performed as previously described . After an = 19) received a single IT injection of Chito-Dota in one ear and Hya-Dota in the contralateral ear. Each animal underwent MRI immediately after injection and on two more occasions over 2 weeks. Anesthetized animals were placed in the supine position with the middle ear cavity situated within the sensitive region of a four-channel phased array coil originally designed for rat heart imaging  within an MR-compatible animal holder. The bed was positioned in the isocenter of a horizontal 9.4 T Agilent magnet equipped with a 12-cm inner diameter gradient system with a maximum gradient strength of 6000 mT/m and an actively tuned birdcage coil . Respiration rate was monitored throughout the experiment, and core body temperature was maintained at 37°C using feedback-controlled warm air . T1-weighted 3D images were acquired with a gradient echo 3D (GE3D) sequence: repetition time, 5.16 ms; echo time, 2.60 ms; number of averages, 4; flip angle, 20°; data matrix, 256 × 256 × 348; field of view (FOV), 38.4 × 38.4 × 57.6 mm3; resolution scan duration, 33 min and 51 s including two dummy scans.As described in section “Study Design”, guinea pigs (The bias field was characterized from the ratio of blurred (1.6% of FOV) 3D images acquired separately with the surface and volume coils using identical parameters as for the high-resolution images, apart from a reduced matrix size of 256 × 192 × 192.After imaging, the animals were sacrificed by an i.p. injection of sodium pentobarbital, followed immediately by decapitation.1  and ITK-snap [version 3.41 ].To investigate effects on hearing thresholds, mixed linear modeling was used to account for heteroscedasticity and autocorrelation in terms of time point, frequency, and IT administration . Frequency was included as a continuous variable. The model with the lowest maximum likelihood and fewest estimated parameters was considered to have the best fit; this was determined with a likelihood ratio chi-square test.Repeated measures analysis of variance (ANOVA) was used to analyze the percentage loss of OHCs in each row  as the within-subjects effect and the two types of IT administration (two levels: Thio-25 and none) as the within-subjects effect.An alpha level of 0.05 was used throughout, and all tests were two-sided. All calculations were performed in IBM SPSS Statistics .F = 146.18, p < 0.0001) and significant interactions between frequency and IT administration , frequency and time point , and IT administration and time point . The estimates of fixed effects are given in The first part of the study investigated conductive hearing loss induced by unilateral IT injection of two sodium thiosulfate-containing particulate chitosan gels, Thio-25 and Thio-40. The hearing thresholds of each animal measured before and after injection are presented in No significant IHC or OHC loss was found in the first five animals of the Thio-25  and ThioF = 56.68, p < 0.0001) and frequency , and significant interactions between time point and IT administration  and between time point and frequency  were found. The fixed effects estimates are given in As the IT administration of Thio-25 induced less conductive hearing loss than Thio-40 and because neither treatment caused hair cell loss, the protective effects of Thio-25 on cisplatin-induced ototoxicity were explored. Electrophysiological hearing thresholds before and 10 days after IT administration of Thio-25 followed by i.v. cisplatin injection are shown in This experiment was repeated using Placebo-25 (the chitosan-based vehicle without sodium thiosulfate) instead of Thio-25. Ten days after cisplatin administration, animals in the Placebo-25 group had symmetrical, bilateral electrophysiological hearing threshold elevations , suggestThe experiment was then repeated a third time, using NaCl instead of Thio-25. Ten days after cisplatin administration, the animals had symmetrical, bilateral electrophysiological hearing threshold elevations . Thus, IF = 35.868, p < 0.001], 2 , and 3 , as well as statistically significant differences in terms of IT administration . The post hoc analysis results are shown in A representative example of the hair cell loss observed in cisplatin-injected animals that were unilaterally pretreated with IT Thio-25 (group Thio-25-cispt) is shown in In the last part of the study, serial MRI was performed to monitor the middle ear distributions of chitosan and hyaluronan, including clearance from the middle ear cavity over a period of 14 days. The polymers were conjugated with a paramagnetic contrast agent to further improve detection and enable estimation of gel clearance over time. Minutes after injection, the contrast-enhanced gel appeared hyperintense and its location in the middle ear cavity was clearly seen, as seen in in vivo study in guinea pigs provides key data relating to the safety, efficacy, and elimination of an IT drug delivery system composed of an antioxidant incorporated in a particulate hydrogel drug carrier. A sodium thiosulfate-containing, chitosan-based system reduced ototoxic injury induced by a single high dose of cisplatin. Cisplatin is a common anticancer agent that predominantly exerts its effects through DNA-dependent mechanisms . In the second part of the study, the effects of the drug carried itself on cisplatin-induced ototoxicity was also investigated. ABR assessment showed no significant difference between vehicle-treated and untreated ears. Therefore, hair cell loss in vehicle-treated ears was not assessed, largely because the technique is very laborious. As chitosan exhibits antioxidant properties  and the Regional Ethical Review Board in Stockholm (No. 138/15).All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.MA is CEO at Flexichem AB, the inventor and current holder of the Viscosan manufacturing process. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer SH declared a shared affiliation, though no other collaboration, with one of the authors PVP to the handling Editor.